Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Construction of genomic phage libraries of the arbuscular mycorrhizal fungi Glomus mosseae and Scutellospora castanea and isolation of ribosomal RNA genes

 Genomic phage libraries of arbuscular mycorrhizal fungi were constructed for the first time, and clones containing ribosomal RNA (rRNA) genes isolated for Glomus mosseae and Scutellospora castanea. The number of rDNA clones per library indicates that these libraries can be also used to isolate genes with low copy numbers. Sequences of the 18S rRNA gene, of the internal transcribed spacer and of the 5.8S rRNA gene were analysed and compared. Differences between the 18S and the 5.8S rRNA genes were few and in the range of variation found for other fungi. In contrast, the internal transcribed spacers of G. mosseae and S. castanea were highly variable, showing the potential of this region for the identification of different species or isolates. Interestingly, nucleotide exchanges were found in this region when the sequence for G. mosseae was compared to those of two other clones of the same isolate. Accepted: 10 November 1995     

Community structure of arbuscular mycorrhizal fungi in undisturbed vegetation revealed by analyses of LSU rDNA sequences

Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with plant roots and are found in most ecosystems. In this study the community structure of AMF in a clade of the genus Glomus was examined in undisturbed costal grassland using LSU rDNA sequences amplified from roots of Hieracium pilosella. Roots were sampled from May to November along eight 30-m transects, 30-120 m apart. Phylogenetic analysis of the sequences revealed 11 phylogenetic clusters within the clade of Glomus. The phylogenetic clusters were patchily distributed within the area; time had no influence on the distribution pattern. The dominant cluster covered up to 10 m along the transect, whereas other clusters formed what can be interpreted as small individual mycelia. Four of the phylogenetic clusters included known species; the other clusters, including the dominant sequence types, were unknown. The dominant phylogenetic cluster enclosed nine haplotypes, and analyses of genetic diversity of this phylogenetic cluster showed that the total diversity could be found within single root fragments, suggesting that the multiple sequences were derived from a single individual.     

Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi‐natural grasslands

Glomus intraradices, an arbuscular mycorrhizal fungus (AMF), is frequently found in a surprisingly wide range of ecosystems all over the world. It is used as model organism for AMF and its genome is being sequenced. Despite the ecological importance of AMF, little has been known about their population structure, because no adequate molecular markers have been available. In the present study we analyse for the first time the intraspecific genetic structure of an AMF directly from colonized roots in the field. A recently developed PCR‐RFLP approach for the mitochondrial rRNA large subunit gene (mtLSU) of these obligate symbionts was used and complemented by sequencing and primers specific for a particularly frequent mtLSU haplotype. We analysed root samples from two agricultural field experiments in Switzerland and two semi‐natural grasslands in France and Switzerland. RFLP type composition of G. intraradices (phylogroup GLOM A‐1) differed strongly between agricultural and semi‐natural sites and the G. intraradices populations of the two agricultural sites were significantly differentiated. RFLP type richness was higher in the agricultural sites compared with the grasslands. Detailed sequence analyses which resolved multiple sequence haplotypes within some RFLP types even revealed that there was no overlap of haplotypes among any of the study sites except between the two grasslands. Our results demonstrate a surprisingly high differentiation among semi‐natural and agricultural field sites for G. intraradices. These findings will have major implications on our views of processes of adaptation and specialization in these plant/fungus associations.     

Suitability of chloroplast LSU rDNA and its diverse group I introns for species recognition and phylogenetic analyses of lichen-forming Trebouxia algae

To date, species identification of lichen photobionts has been performed principally on the basis of microscopic examinations and molecular data from nuclear-encoded genes. In plants, the chloroplast genome has been more readily exploited than the nuclear genome for systematic investigations. At the present time, very little information is available about the chloroplast genome of lichen-forming algae. For this reason, we have sequenced a portion of the gene encoding for the chloroplast large sub-unit rRNA (LSU rDNA) as a new molecular marker. Sequencing of the chloroplast LSU rDNAs revealed the existence of an unusual diversity of group I introns (a total of 31) within 15 analyzed Trebouxia species. The number, sequence and insertion site of these introns were very different among species, contributing to their recognition. A relatively large intron-free portion of the chloroplast LSU rDNA and part of the nuclear ribosomal cistron (18S–5.8S–26S) between the nuclear internal transcribed spacers (nrITS) were subjected to phylogenetic analyses. The obtained results indicate that data combination from both nuclear and chloroplast sequences can improve phylogenetic accuracy. Herein, we propose the suitability of both intronic and exonic sequences of the chloroplast LSU rDNA for species recognition, and an exonic sequence spanning from position 879 to 1837 in the Escherichia coli 23S rDNA for phylogenetic analyses of Trebouxia phycobionts.     

Evolution and selection of Rhg1, a copy‐number variant nematode‐resistance locus

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2‐kb unit contains four genes. One allele of Rhg1, Rhg1‐b, is responsible for protecting most US soybean production from SCN. Whole‐genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2‐kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high‐density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non‐neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1.     

Eu-Chlorella large subunit rDNA sequences and group I introns in ribosomal DNA of the paramecian symbiotic alga NC64A

Although the examination of large subunit ribosomal RNA genes (LSU rDNA) is advanced in phylogenetic studies, no corresponding sequence data from trebouxiophytes have been published, with the exception of ‘Chlorella’ellipsoidea Gerneck. We determined the LSU rDNA sequence of Chlorella vulgaris Beijerinck and of the symbiotic alga of green paramecium, Chlorella sp. NC64A. A total of 59 nucleotide substitutions were found in the LSU rDNA of the two species, which are disproportionately distributed. Primarily, 65% of the substitutions were encountered in the first 800 bp of the alignment. This segment apparently has evolved eight times faster than the complete SSU rDNA sequence, making it a good candidate for a phylogenetic marker and giving a resolution level intermediate between small subunit (SSU) rDNA and internal transcribed spacers. Green algae are known as a group I intron‐rich group along with rhodophytes and fungi. NC64A is particularly rich in the introns; five introns were newly identified from the LSU rDNA sequence, which we named Cnc.L200, Cnc.L1688, Cnc.L1926, Cnc.L2184 and Cnc.L2437, following the insertion positions. In the present study we analyzed these introns with three others (Cnc.S943, Cnc.S1367 and Cnc.S1512) that had already been found in NC64A SSU rDNA. Secondary structure modeling placed these introns in the group I intron family, with four introns belonging to subgroup C1 and the other four introns belonging to subgroup E. Five of the intron insertion positions are unique to the paramecian symbiont, which may indicate relatively recent events of intron infections that includes transpositions. Intron phylogeny showed unprecedented relationships; four Cnc. IC1 introns made a clade with some green algal introns with insertions at nine different positions, whereas four Cnc. IE introns made a clade with the S651 intron (Chlorella sp. AN 1–3), which lay as a sister to the S516 insertion position subfamily.     

MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Spatial soil heterogeneity has a greater effect on symbiotic arbuscular mycorrhizal fungal communities and plant growth than genetic modification with Bacillus thuringiensis toxin genes

Maize, genetically modified with the insect toxin genes of Bacillus thuringiensis (Bt), is widely cultivated, yet its impacts on soil organisms are poorly understood. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots and may be uniquely sensitive to genetic changes within a plant host. In this field study, the effects of nine different lines of Bt maize and their corresponding non‐Bt parental isolines were evaluated on AMF colonization and community diversity in plant roots. Plants were harvested 60 days after sowing, and data were collected on plant growth and per cent AMF colonization of roots. AMF community composition in roots was assessed using 454 pyrosequencing of the 28S rRNA genes, and spatial variation in mycorrhizal communities within replicated experimental field plots was examined. Growth responses, per cent AMF colonization of roots and AMF community diversity in roots did not differ between Bt and non‐Bt maize, but root and shoot biomass and per cent colonization by arbuscules varied by maize cultivar. Plot identity had the most significant effect on plant growth, AMF colonization and AMF community composition in roots, indicating spatial heterogeneity in the field. Mycorrhizal fungal communities in maize roots were autocorrelated within approximately 1 m, but at greater distances, AMF community composition of roots differed between plants. Our findings indicate that spatial variation and heterogeneity in the field has a greater effect on the structure of AMF communities than host plant cultivar or modification by Bt toxin genes.     

The draft genome of a wild barley genotype reveals its enrichment in genes related to biotic and abiotic stresses compared to cultivated barley

Wild barley (Hordeum spontaneum) is the progenitor of cultivated barley (Hordeum vulgare) and provides a rich source of genetic variations for barley improvement. Currently, the genome sequences of wild barley and its differences with cultivated barley remain unclear. In this study, we report a high‐quality draft assembly of wild barley accession (AWCS276; henceforth named as WB1), which consists of 4.28 Gb genome and 36 395 high‐confidence protein‐coding genes. BUSCO analysis revealed that the assembly included full lengths of 95.3% of the 956 single‐copy plant genes, illustrating that the gene‐containing regions have been well assembled. By comparing with the genome of the cultivated genotype Morex, it is inferred that the WB1 genome contains more genes involved in resistance and tolerance to biotic and abiotic stresses. The presence of the numerous WB1‐specific genes indicates that, in addition to enhance allele diversity for genes already existing in the cultigen, exploiting the wild barley taxon in breeding should also allow the incorporation of novel genes. Furthermore, high levels of genetic variation in the pericentromeric regions were detected in chromosomes 3H and 5H between the wild and cultivated genotypes, which may be the results of domestication. This H. spontaneum draft genome assembly will help to accelerate wild barley research and be an invaluable resource for barley improvement and comparative genomics research.     

Comparative DNA sequence analyses of Pyramimonas parkeae (Prasinophyceae) chloroplast genomes

Prasinophytes form a paraphyletic assemblage of early diverging green algae, which have the potential to reveal the traits of the last common ancestor of the main two green lineages: (i) chlorophyte algae and (ii) streptophyte algae. Understanding the genetic composition of prasinophyte algae is fundamental to understanding the diversification and evolutionary processes that may have occurred in both green lineages. In this study, we sequenced the chloroplast genome of Pyramimonas parkeae NIES254 and compared it with that of P. parkeae CCMP726, the only other fully sequenced P. parkeae chloroplast genome. The results revealed that P. parkeae chloroplast genomes are surprisingly variable. The chloroplast genome of NIES254 was larger than that of CCMP726 by 3,204 bp, the NIES254 large single copy was 288 bp longer, the small single copy was 5,088 bp longer, and the IR was 1,086 bp shorter than that of CCMP726. Similarity values of the two strains were almost zero in four large hot spot regions. Finally, the strains differed in copy number for three protein‐coding genes: ycf20, psaC, and ndhE. Phylogenetic analyses using 16S and 18S rDNA and rbcL sequences resolved a clade consisting of these two P. parkeae strains and a clade consisting of these plus other Pyramimonas isolates. These results are consistent with past studies indicating that prasinophyte chloroplast genomes display a higher level of variation than is commonly found among land plants. Consequently, prasinophyte chloroplast genomes may be less useful for inferring the early history of Viridiplantae than has been the case for land plant diversification.     

Genetic variation and gene conversions within the bovine NK‐lysin gene family

In contrast to a single copy of the NK‐lysin gene in humans and many other mammals, we previously identified a family of four expressed NK‐lysin genes arising by tandem duplications on cattle chromosome 11. Here, we report two genetic variants in the bovine NK‐lysin complex with potential importance in the bovine innate immune system. The first one is a 9‐bp deletion causing a three‐amino‐acid deletion in the pro‐region of the NK1 gene product. The second is a deletion of NK2B in some Holstein cattle, resulting in copy number variation that is in disequilibrium with a SNP from the bovine 770K HD SNP array. We also show evidence for gene conversions within the three new NK2 genes, which at least partially accounts for their high degree of sequence identity.     

Chromosome‐specific physical localisation of expressed sequence tag loci in Corchorus olitorius L.

Jute (Corchorus spp.), as a natural fibre‐producing species, ranks next only to cotton. Inadequate understanding of its genetic architecture is a major lacuna for genetic improvement of this crop in terms of yield and quality. Establishment of a physical map provides a genomic tool that helps in positional cloning of valuable genes. In this report, an attempt was initiated to study association and localisation of single copy expressed sequence tag (EST) loci in the genome of Corchorus olitorius. The chromosome‐specific association of EST was determined based on the appearance of an extra signal for a single copy cDNA probe in mitotic interphase nuclei of specific trisomic(s) for fluorescence in situ hybridisation, and validated using a cDNA fragment of the 26S rRNA gene (600 bp) as molecular probe. The probe exhibited three signals in meiotic interphase nuclei of trisomic 5, instead of two as observed in diploids and other trisomics, indicating its association with chromosome 5. Subsequent hybridisation of the same probe on the pachytene chromosomes of diploids confirmed that 26S rRNA occupies the terminal end of the short arm of chromosome 5 in C. olitorius. Subsequently, chromosome‐specific association of 63 single copy EST and their physical localisation were determined on chromosomes 2, 4, 5 and 7. The study describes chromosome‐specific physical localisation of genes in jute. The approach used here could be a step towards construction of genome‐wide physical maps for any recalcitrant plant species like jute.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.